OPIOID PEPTIDE RECEPTOR STUDIES .3. INTERACTION OF OPIOID-PEPTIDES AND OTHER DRUGS WITH 4 SUBTYPES OF THE KAPPA(2) RECEPTOR IN GUINEA-PIG BRAIN

Using guinea pig, rat, and human brain membranes depleted of mu and de lta receptors by pretreatment with the site-directed acylating agents BIT (mu selective) and FIT (delta selective), previous studies from ou r laboratory resolved two subtypes of the kappa(2) binding site, terme d kappa(2a) and kappa(2b). In more recent studies, we used 6 125)Iodo] -3,14-dihydroxy-17-cyclopropyylmethyl-4,5 alpha-epoxymorphinan ([I-125 ]IOXY) to characterize multiple kappa(2) binding sites in rat brain. T he results indicated that [I-125]IOXY, like [H-3]bremazocine, selectiv ely labels kappa(2) binding sites in rat brain membranes pretreated wi th BIT and FIT. In the rat brain, using 100 nM [D-Ala(2)-MePhe(4),Gly- ol(5)] enkephalin to block [I-125]IOXY binding to the kappa(2b) site, we resolved two subtypes of the kappa(2a) binding site. In the present study we examined the binding of [I-125]IOXY to the kappa(2) receptor s of guinea pig brain. As observed in rat brain, [I-125]IOXY, under ap propriate assay conditions, selectively labels kappa(2) binding sites. Quantitative binding studies readily demonstrated the presence of kap pa(2a) and kappa(2h) binding sites. The kappa(2a) binding sites were s electively assayed using 5 mu M [Leu(5)]enkephalin to block [I-125]IOX Y binding to the kappa(2a) sites, and kappa(2b) sites were selectively assayed using 5 mu M (-)-(1S,2S)-U50,488 to block [I-125]IOXY binding to the kappa(2a) sites. Under these conditions, two subtypes of the k appa(2a) site were resolved with high (kappa(2a-1)) and low (kappa(2a- 2)) affinity for nor-BNI (K-i values = 0.88 and 476 nM) and CI977 (K-i values = 17.5 and 95,098 nM). Similarly, two subtypes of the kappa(2b ) site were observed with high (kappa(2h-1)) and low (kappa(2b-2)) aff inity for [D-Ala(2)-MePhe(4),Gly-ol(5)]enkephalin (DAMGO) (K-i values = 97 and 12,321 nM) and alpha-neoendorphin (K-i values = 33 and 5308 n M). Two-site models were also resolved in the presence of 100 mu M 5'- guanylyimidodiphosphate (GppNHp). We carried out detailed ligand selec tivity analysis of the multiple kappa(2) binding sites. Most test agen ts were either nonselective or selective for the kappa(2a-2) site. Nal buphine was moderately selective for the kappa(2a-2) site. Similarly, although most test agents were either nonselective or selective for th e kappa(2b-1) site, butorphanol, and the delta antagonists naltrindole , naltriben, and 7-benzylidene-7-dehydronaltrexone were moderately sel ective for the kappa(2b-2) site. Of the endogenous opioid peptides tes ted, BAM22P had the highest affinity for the kappa(2b-2). Site (31 nM) and peptide E had the highest affinity for the kappa(2a-2) binding si te (53 nM). These data provide additional evidence for heterogeneity o f the kappa-opioid receptor and new targets for drug design, synthesis , and therapeutics.